We propose to use a canine model for the study of acute ischemia in which alterations of regional blood flow can be quantified using radioactive microspheres. This model enables highly reproducible estimation of myocardial blood flow in normal and ischemic animals for the analysis of perturbations of myocardial lipids, membrane-bound enzymes, and myocardial organelles (lysosomes, microsomes, mitochondria, and sarcolemma). We propose to extend this approach to isolated adult canine myocytes from control and ischemic myocardium. Isolated myocytes will be studied for alterations in lipids, enzymes and specialized functions of organelles (e.g., lysosomal latency, Ca2 ions uptake by sarcoplasmic reticulum, mitochondrial oxidative phosphorylation, and sarcolemmal permeability). Attempts will be made to simulate conditions of ischemia in vitro (e.g. acidosis, high Ca2 ion, etc.) using these isolated cells. The nature of the molecular mechanism of membrane injury in ischemia of the myocardium remains a critical area of basic research. Exogenous phospholipases can alter membrane permeability and inactivate critical membrane-bound enzymes; the production of lysophospholipids and free fatty acids may be critical in this lipolytic injury. The loss of membrane integrity may be influenced by activation of myocardial lipases during ischemia. We propose to assess in situ parameters of lipolytic activation as well as the susceptibility of membrane of the myocardium to lipase-induced injury in vitro.